In a breast-fed infant, bifidobacteria (bacteria of genus Bifidobacterium) accounts for 80 to 90% of intestinal bacterial flora, and is most predominant. Bifidobacteria is known to maintain the intestinal tract function of an infant and in the intestinal tract to prevent infections of putrefactive bacteria invading from the outside and opportunistic infection due to amplification of indigenous bacteria which usually exist in the intestinal tract and are harmless but become putrefactive when the infant is in a poor physical condition. In the field of the infant nutrition, it has been designed to prevent an infective disease or to promote treatment of an allergic disease by adding infant-type bifidobacteria to an infant formula or administering formulated bifidobacteria. For such use, from a physiological viewpoint, bifidobacteria existing in a human body is preferably administered to a human being, and human-type bifidobacteria is desirably used. Examples of known bifidobacteria existing in infant feces include Bifidobacterium infantis and Bifidobacterium breve. Of those, Bifidobacterium infantis is bifidobacteria detected only in an infant.
In order to evaluate the efficiency of such bifidobacteria application, for example, it is necessary to calculate the existing population of bifidobacteria in feces, and moreover, to judge a clinical effect by determining the individual species of Bifidobacterium. Accordingly, a technique for detecting Bifidobacterium infantis with reliability has been required.
Conventionally, bifidobacteria in feces or environmental substances has been identified by culturing a specimen under an aerobic condition, isolating colonies and performing a morphological test such as gram staining, observation of the shape of bacteria by microscopic examination or a biochemical test such as the determination of fructose-6-phosphate phosphoketolase activity, oxygen tolerance, or fermentation pattern of carbohydrate. The aforementioned specimen is cultured according to a standard method that is generally used for measurement of viable count or identification of bacterial species, but this approach has many problems. For example, not all bifidobacteria in feces can always be cultured. Moreover, the culture is time-consuming and troublesome because bifidobacteria must be cultured by using an anaerobic apparatus, and the culture requires about a few days to a week. Furthermore, bifidobacteria in a specimen must be stored anaerobically and at a low temperature and be subjected to analysis in the condition of viable cells, and much skill was required for the analysis.
On the other hand, recently, for identifying bifidobacteria, the similarity with a reference strain has been determined by the DNA-DNA homology test or by analysis of a nucleotide sequence of 16S rRNA gene. The DNA-DNA homology test is a method including culturing isolated bacteria to be tested, preparing the chromosomal DNA of the bacteria, and comparing it with that of a reference strain for homology by hybridization (Non-Patent Document 1). In order to prepare chromosomal DNA necessary for the test, the absolute requirement for the method is that the bacteria should be culturable. In addition, even if the bacteria are culturable, the method is troublesome because the chromosomal DNA must be prepared in large quantities. Furthermore, the method has, for example, an essential problem in that the method is suitable for examination of bacteria that have a far genetic distance from each other, but it is not sensitive for determining that of related species.
16S rRNA gene generally exists in prokaryotes, and there are nucleotide sequences conserved in different genus of bacteria and sequences conserved in different species of bacteria, so that it can be used for identification of bacterial species. The method can be performed by determining the nucleotide sequence of 16S rRNA gene of test bacteria, aligning the nucleotide sequence with a sequence of 16S rRNA gene of related species of bacteria (alignment), and analyzing the difference of the nucleotide sequences. However, the method needs to determine, as a reference, the sequence of 16S rRNA gene of bacteria existing in soil, sludge, feces, foods, or the like, and to analyze many sequences systematically, so that the method is time-consuming and troublesome (Non-Patent Document 2).
Moreover, use of a probe for detecting a specific sequence of 16S rRNA gene allows one to determine whether or not the test bacteria are the same bacteria as reference bacteria of which 16S rRNA gene has a known specific sequence. As an example of applying such a technique, a detection method using an oligonucleotide and derivatives thereof as probes for detecting 16S rRNA gene of Bifidobacterium infantis as a target is disclosed (Patent Document 1). However, it is a problem that the method is time-consuming. On the other hand, a technique in which bifidobacteria is analyzed using PCR primers capable of detecting 16S rRNA gene is known (Patent Document 2 and Non-Patent Document 3). However, the primers that are disclosed in those documents as specific primers for Bifidobacterium infantis have a problem of detecting Bifidobacterium breve in error.    Patent Document 1: JP 06-197763 A    Patent Document 2: JP 11-123093 A    Non-Patent Document 1: Bergey's Manual of Systematic Bacteriology Vol. 2 p1418-1434 (1986)    Non-Patent Document 2: Progress in Nucleic Acid Research and    Molecular Biology Vol. 32 p155-216 (1985)    Non-Patent Document 3: Systematic and Applied Microbiology Vol. 25 p. 536 (2002)